Abstract Hematopoietic stem cells (HSCs) are capable of self-renewal and multi-lineage differentiation to maintain lifelong production of all blood cells. However, the regenerative capacity of HSCs declines with age, and old HSCs exhibit a skewed differentiation towards myeloid lineages. These age-associated changes are responsible for decreased immunity and increased propensity for anemia and myeloid malignancies in the elderly. To gain insight into the molecular mechanisms of age-associated decline in HSC function, we propose to investigate the roles of microRNAs (miRNAs) in HSC self-renewal, differentiation and aging. Recent studies have found a miRNA expression pattern in HSCs that is different from those in the differentiated hematopoietic lineages, suggesting that a distinctive set of miRNAs regulate the function of HSCs. However, the majority of miRNAs expressed in HSCs have not been studied for their regulation of HSC self-renewal and differentiation. Furthermore, little is known about whether these miRNAs are responsible for HSC aging. We hypothesize that the ability of HSCs to self-renew and differentiate is regulated by a set of miRNAs, and deletion of these miRNAs leads to altered HSC self-renewal and differentiation. We further hypothesize that certain miRNAs that negatively regulate the self-renewal and reconstitution capacity of HSCs represent ?aging? miRNAs, and deletion of the critical ?aging? miRNAs can rejuvenate old HSCs and reverse HSC aging. We will test these hypotheses with the following two Aims. In Aim 1, we will use an unbiased CRISPR-based genetic screen to identify miRNAs that regulate HSC self- renewal and differentiation. A lentiviral CRISPR library with 1003 single-guide RNAs (sgRNAs) that target 230 miRNA expressed in mouse HSCs will be used to infect HSCs from young mice expressing Cas9. Infected HSCs that carry different miRNA-targeting sgRNAs will be injected into stem cell-depleted recipient mice to repopulate competitively with each other in successive transplantations. Altered HSC self-renewal and function will lead to either enrichment or depletion of HSCs. By analyzing the abundance of sgRNAs in HSCs and differentiated B, T or myeloid lineages using deep sequencing before and after transplantations, we will identify the corresponding miRNAs that positively or negatively regulate HSC self-renewal and differentiation. In Aim 2, we will test whether it is possible to rejuvenate old HSCs by CRISPR targeting of 4 miRNAs (miR-126, miR-132, miR-193b and miR-212) that have been shown to negatively regulate HSC self-renewal in young mice (2-4). Furthermore, we will prioritize and select candidate miRNAs identified in Aim 1 for validation based on their significant enrichment or depletion in the screen. We will determine whether the processes affected by these miRNAs involve HSC homing, proliferation, quiescence or differentiation. Once being validated, selected miRNA will be studied for their expression during aging and abilities to rejuvenate old HSCs. The proposed studies will provide novel insights into the underlying mechanisms of HSC self-renewal, differentiation and aging, and establish a paradigm for the translational potential of HSC rejuvenation to ameliorate age-associated diseases and promote healthspan.